Methods for use of mpl ligands with primitive human stem cells

ABSTRACT

Myeloproliferative leukemia receptor (mpl) ligands, such as thrombopoietin, act on a primitive subpopulation of human stem cells having the characteristics of self-renewal and ability to give rise to all hematopoietic cell lineages. Thrombopoietin supports both megakaryocytic differentiation and primitive progenitor cell expansion of CD34 +  and CD34 +  sub-populations (CD34 + Lin − , CD34 + Thy-1 + Lin − , and CD34 + Lin −  Rh123 lo ). Thrombopoietin also stimulated quiescent human stem cells to begin cycling. Thus, mpl ligands are useful for expanding primitive stem cells for restoration of hematopoietic capabilities and for providing modified human stem cells for gene therapy applications.

[0001] This is a continuation of application Ser. No. 09/328,188, filedJun. 8, 1999, now U.S. Pat. No. 6,326,205, which is a divisional ofapplication Ser. No. 08/550,167, filed Oct. 30, 1995, now U.S. Pat. No.6,060,052, the disclosures of which are incorporated herein byreference.

FIELD OF THE INVENTION

[0002] This invention relates to myeloproliferative receptors (mpl),specifically, the use of mpl ligands to expand primitive human stem cellsubpopulations with minimal differentiation.

BACKGROUND OF THE INVENTION

[0003] Thrombopoietin (TPO) is a recently isolated ligand of mpl(Bartley et al. (1994) Cell 77:1117; Kaushansky et al. (1994) Nature369:568; Lok et al. (1994) Nature 269:565; Kuter et al. (1994) Proc.Natl. Acad. Sci. USA 91:11104-11108; Kuter & Rosenberg (1994) Blood84:1464; Wendling et al. (1994) Nature 369:571), first identified as theproto-oncogene transduced by the murine myeloproliferative leukemia(MPL) virus (Wendling et al. (1989) Blood 73:1161-1167; Souyri et al.(1990) Cell 63:1137-1147; Vigon et al. (1992) Proc. Natl. Acad. Sci. USA89:5640-5644; Skoda et al. (1993) EMBO J. 12:2645-2653; Methia et al.(1993) Blood 82:1395-1401). TPO has been shown to independentlystimulate megakaryocyte (MK) progenitor division and MK maturation invivo and in vitro (Bartley et al. (1994) supra; Kuter et al. 1994)supra; Kuter & Rosenberg (1994) supra; Wendling et al. (1994) supra; deSauvage et al. (1994) Nature 369:533; Broudy et al. (1995) Blood85:1719-1726; Lok & Foster (1994) Stem Cells 12:586-598; Zeigler et al.(1994) Blood 84:4045). In vivo administration of TPO to thrombocytopenicrodents was found to significantly boost the platelet count as well asincrease the number and ploidy of maturing MKs in the bone marrow (Loket al. (1994) supra; Lok et al. (1994) supra; Wendling et al. (1994)supra; de Sauvage et al. (1994) supra). Absence of a c-mpl (TPOreceptor) gene in mice was reported to result in thrombocytopenia(Guerney et al. (1994) Science 265:1445). More recently, it has beendemonstrated that MKs can be primed to produce functional platelets inculture after exposure to TPO (Choi et al. (1995) Blood 85:402).

[0004] Hematopoietic stem cells are rare cells that have been identifiedin fetal bone marrow, umbilical cord blood, adult bone marrow, andperipheral blood, which are capable of differentiating into each of themyeloerythroid (red blood cells, granulocytes, monocytes), megakaryocyte(platelets) and lymphoid (T-cells, B-cells, and natural killer cells)lineages. In addition, these cells are long-lived, and are capable ofproducing additional stem cells, a process termed self-renewal. Stemcells initially undergo commitment to lineage restricted progenitorcells, which can be assayed by their ability to form colonies insemisolid media. Progenitor cells are restricted in their ability toundergo multi-lineage differentiation and have lost their ability toself-renew. Progenitor cells eventually differentiate and mature intoeach of the functional elements of the blood. This maturation process isthought to be modulated by a complex network of regulatory factorsincluding erythropoietin (EPO), granulocyte colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),thrombopoietin (TPO), steel factor (Stl), Flk-2 ligand and interleukins(IL) 1-15.

[0005] Recently, in vitro assays have been developed to identify humanhematopoietic stem cells having self-renewal and multi-lineagedifferentiative capacity. One assay is the cobblestone area-forming cell(CAFC) assay, based on freshly isolated stromal cells or establishedstromal cell lines. In the mouse system, late-appearing cobblestone areaformation on fresh marrow-derived stroma (Ploemacher et al. (1991) Blood78:2527) or on a cloned stromal cell line (Neben et al. (1993) Exp.Hematol. 21:438) has been shown to correlate with in vivo hematopoieticrepopulating ability. Correlation of CAFC and long termculture-initiating cell (LTCIC) frequencies using the mouse stromal cellline Sysi has been demonstrated (Reading et al. (1994) Exp. Hematol.22:406). In addition, the in vivo severe combined immunodeficiency(SCID)-hu bone assay has been used to measure long term engraftment ofcandidate stem cell populations (Kyoizumi et al. (1992) Blood 79:1704;Baum et al. (1992) Proc. Natl. Acad. Sci. USA 89:2804; Chen et al.(1993) Blood 82 (Suppl. 1):180a). Both the in vitro CAFC assay and theSCID-hu bone model permit analysis of B-cell and myeloid cell generationfrom candidate pluripotent hematopoietic stem cells (PHSC).

[0006] It is becoming increasingly apparent that distinct subpopulationsof stem cells may be responsible for different phases of engraftmentpost transplantation. As early as 1964, differences in the ability ofmurine spleen colony forming units (CFU-S) to generate secondary CFU-Swere defined (Ploemacher & Brons (1994) Exp. Hematol. 17:263-266).Although evidence now indicates that most CFU-S are not involved inrepopulating lethally irradiated hosts (Jones et al. (1990) Nature347:188-189; Jones et al. (1989) Blood 73:397-401), heterogeneity intransplantation potential appears to exist even within subpopulations ofradioprotective cells. This has been demonstrated with serial bonemarrow transplantations. The long-term repopulating ability of thegrafts are lost with serial transfers, while a cell population surviveswhich contributes to short-term reconstitution (Jones et al. (1989)supra). Further support for the concept that both short-term andlong-term reconstituting stem cell populations exist have been derivedfrom studies in which isoenzyme analysis and retroviral gene marking ofhematopoietic cells have been utilized to track the fate of stem cells.A mathematical analysis of correlations and variances of donorreconstitution with isoenzyme variants in lethally irradiated miceindicates that a large number of multi-lineage clones are activeimmediately after reconstitution but rapidly decline, with the majoritybeing inactive 12 weeks post-transplantation (Harrison & Zhong (1992)Proc. Natl. Acad. Sci. USA 89:10134-10138; Harrison et al. (1993) Exp.Hematol. 21:206-219). These observations indicate the existence of apopulation of cells with multi-lineage short-term engrafting potentialin donor murine bone marrow. Similar observations have been made in alarge animal transplantation model, where isoenzyme differences haveindicated the contribution of multiple clones to short-term engraftmentfollowed by sustained contributions by relatively few stem cell clones(Abkowwitz et al. (1990) Proc. Natl. Acad. Sci. USA 87:9062-9066). Thesefindings have been confirmed by an eloquent analysis of clonaldevelopment after transplantation with retrovirally marked stem cells(Jordan et al. (1990) Cell 61:953; Capel et al. (1990) Blood 75:2267).

[0007] Theoretically, subsets of cells with differing proliferativepotentials may also differ with regards to physical characteristics, andtherefore may be isolated and functionally defined. Bone marrow cellsresponsible for reconstitution following lethal irradiation can beisolated using centrifugation techniques exploiting cell size anddensity fractionation, or fluorescence-activated cell sorting (FACS)based on uptake of fluorescent vital dyes, lectin binding, or cellsurface antigen expression (Sprangrude (1989) Immunol. Today 10:344-350;Visser & Van Bekkum (1990) Exp. Hematol. 18:248-256). FACS isolatedmurine cells that are responsible for engraftment lack lineage markersfor B-cells, T-cells, myelomonocytes and erythrocytes and are termedlineage negative (Lin⁻) (Spangrude et al. (1988) Science 241:58-62). TheLin⁻ fraction of murine bone marrow can be further subdivided based onlow levels of Thy-1 and expression of the Sca-1 antigen (Visser & VanBekkum (1990) supra; Spangrude et al. (1988) supra; Szilvassay et al.(1989) Blood 74:930-939; Szilvassay & Cory (1993) Blood 81:2310-2320;Spangrude & Scollay (1990) Exp. Hematol. 18:9920-926; Jurecic et al.(1993) Blood 82:2673-2683). Murine cells that are Thy-1.1^(lo)Lin⁻Sca-1⁺are 1000-fold enriched in radioprotective ability, and contain all ofthe radioprotective cells found in the bone marrow of syngeneicC57BL/thy-1.1 mice (Uchida & Weissman (1992) J. Exp. Med. 175:175-184).As few as 100 cells that are Thy-1.1^(lo) Lin⁻Sca-1⁺ can radioprotect atleast 95% of lethally irradiated recipients with long term donor derivedreconstitution. At this cell dose, Thy-1.1^(lo)Lin³¹ Sca-1⁺ cells giverise to donor peripheral blood white blood cells by 10 days posttransplant, and to platelets within 14 days of transplant (Uchida et al.(1994) Blood 83:3758-3779). These studies suggest this populationcontains cells with both short-term and long-term engrafting potential.

[0008] Thy-1.1^(lo)Lin⁻Sca-1⁺ cells can be further divided byheterogeneity of cell cycle status and uptake of a fluorescent dye,rhodamine 123 (Spangrude & Johnson (1990) Proc. Natl. Acad. Sci. USA87:7433-7437; Li & Johnson (1992) J. Exp. Med. 175:1443-1447; Fleming etal. (1993) J. Cell Biol. 122:897-902; Wolf et al. (1993) Exp. Hematol.21:614-622). Rhodamine 123 is a dye that identifies active mitochondria,and its efflux from the cell is handled by the multidrug resistance geneproduct, P-glycoprotein. Those cells that retain small quantities ofrhodamine 123 are termed rhodamine dull (or low) and have been shown topossess marrow repopulating ability (MRA) (Li & Johnson (1992) supra).

[0009] The cells responsible for reconstituting hematopoiesis in humansreceiving a bone marrow transplant reside in a subset of cellsexpressing the CD34 antigen (CD34⁺) (Berenson et al. (1991) Blood77:1717-1722). This Xfraction of cells can be further subdivided basedon multiple antigen characteristics (Lansdorp et al. (1990) J. Exp. Med.172:363-366; Verfaille et al. (1990) J. Exp. Med. 172:509-520; Briddellet al. (1992) Blood 79:3159-3167) including the lack of lineage specificmarkers (Lin⁻) and expression of the Thy antigen (Thy-1⁺) (Baum et al.(1992) Proc. Natl. Acad. Sci. USA 89:2804-2808; Craig et al. (1993) J.Exp. Med. 177:1331-1342; Murray et al. (1990) Blood Cells 20:364-370;Murray et al. (1995) Blood 85:468). In vivo assays using adult bonemarrow and mobilized peripheral blood cells that are CD34⁺Thy-1⁺Lin⁻have shown that this population contains cells capable of contributingto all hematopoietic lineages (Chen et al. (1994) Blood 84:2497-2505;Galy et al. (1994) 84:104-110).

[0010] Mpl expression has been detected by polymerase chain reaction(PCR) in human hematopoietic cells throughout the MK lineage, as well asin primitive CD34⁺CD38^(lo/−) cells (Debili et al. (1995) Blood85:391-401). However, little is known about the actions of TPO onprimitive cells prior to commitment to the MK lineage. One report hasshown that primitive mouse HSC of the phenotype Sca-1⁺AA4⁺ express mpl,and exposure of these cells to TPO in vitro resulted in MKdifferentiation (Zeigler et al. (1994) supra).

SUMMARY OF THE INVENTION

[0011] The present invention is based in part on the discovery thathuman stem cells respond to ligands which bind and activate themyeloproliferative leukemia receptor (mpl) by expansion of primitivenon-megakaryocytic blast cells, as well as expansion of megakaryocytic(MK) progenitor cells, commitment to the MK lineage, and MK maturation.These discoveries provide the basis for new uses, including therapeuticuses of mpl ligands for in vitro and in vivo stem cell activation andexpansion.

[0012] The subset of primitive human stem cells described and disclosedherein to express mpl and respond to a mpl ligand, thrombopoietin (TPO),have the ability to reconstitute hematopoiesis in humans receiving abone marrow transplant. The stem cells useful in this invention arecharacterized by their ability to undergo substantial self-renewal andto proliferate and differentiate into cells of all the hematopoieticlineages. These cells may be identified by cell surface markers such asthe CD34 antigen (CD34⁺). More preferably, the stem cells useful in theinvention are enriched for the ability to undergo substantialself-renewal and to proliferate and differentiate into cells of all thehematopoietic lineages. A more enriched subpopulation of cells may beidentified by the lack of lineage specific markers (Lin⁻), and/orexpression of the Thy-1 antigen (Thy-1⁺) (Baum et al. (1992) supra;Craig et al. (1993) supra). e.g., CD34⁺Lin⁻ or CD34⁺Thy-1⁺Lin⁻. Thesecells are highly enriched in pluripotent cells with long-term andshort-term repopulating potential. Alternatively, an enrichedsubpopulation of cells with the desired characteristics may beidentified by the cell markers CD34⁺Lin⁻Rho^(lo) orCD34⁺Thy-1⁺Lin⁻Rho^(lo). The subpopulations of stem cells useful in thisinvention may also be identified by their light scatteringcharacteristics, e.g., low side scatter and low-to-medium forwardscatter profiles by FACS analysis, and/or by phenotype, e.g., having asize between mature lymphoid cells and mature granulocytes.

[0013] The present invention features a method for promoting thesurvival of long-term repopulating human stem cells by culturing suchcells in the presence of a mpl ligand. The mpl ligand of the inventionis characterized by the ability to bind mpl such that mpl-mediatedbiological activity is initiated. In a preferred embodiment, the mplligand is TPO, more preferably, human TPO, and still more preferably,recombinant human TPO. Stem cells cultured in the presence of a mplligand (e.g., TPO) retain the ability to undergo substantialself-renewal and to proliferate and differentiate into cells of all thehematopoietic lineages, i.e., are pluripotent hematopoietic stem cells.

[0014] The invention features the use of a mpl ligand on a primitivesubpopulation of human stem cells to expand the population of stem cellswith minimal concomitant induction of differentiation. The expanded cellpopulation is characterized by the ability to generate cells capable ofsubstantial self-renewal and proliferation and differentiation intocells of all of the hematopoietic lineages.

[0015] The invention features a therapeutic method for restoringhematopoietic capability to a human subject. Stem cells are purifiedfrom cells removed from a subject, expanded by in vitro exposure to ampl ligand, and returned to the subject, resulting in restoration ofhematopoietic capability to the subject. In addition to expansion in thepresence of the mpl ligand thrombopoietin, in vitro expansion of stemcells can be conducted in the presence of additional cytokines,including for example interleukin-3 (IL-3), leukemia inhibitory factor(LIF), and steel factor (Stl) (also known as c-kit ligand (KL) or stemcell factor (SCF)), interleukin 6 (IL-6), fetal liver tyrosine kinase2/3 (Flk2/Flt3), macrophage inhibitory protein-1α (MIP-1α),granulocyte-macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), IL-1, and IL-11.

[0016] The invention features a method for activating quiescent stemcells to divide by exposing such cells to a mpl ligand. This aspect ofthe invention has important clinical implications, including improvedtransduction of hematopoietic cells by a retroviral vector for use ingene therapy.

[0017] Accordingly, in a related aspect, quiescent stem cells areactivated in the presence of a mpl ligand, and cultured with aretroviral vector containing a gene of interest. The actively dividingcells integrate the gene of interest, and express the foreign geneproduct. Such transformed stem cells are useful for gene therapyapplications.

[0018] In the gene therapy aspect of the invention, hematopoietic cellsare removed from a subject, transduced in vitro in the presence of a mplligand and a retroviral vector, and the modified cells returned to thesubject. The modified stem cells and their progeny will express thedesired gene product in vivo, thus providing sustained therapeuticbenefit.

[0019] In addition to the advantages of stem cell expansion with minimaldifferentiation and stem cell activation, other advantages and featuresof the present invention will become apparent to those skilled in theart upon reading this disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1A is a graph showing the proliferative capacity ofCD34⁺Thy-1⁺Lin⁻ cells purified from adult bone marrow (ABM) and culturedindividually in the presence of 10% supernatant from COS cellstransfected with an empty expression vector (control). Each barrepresents the maximum proliferation achieved by each cell plated over afour weeks culture period, as determined from each series of bi-weeklyimages.

[0021]FIG. 1B is a graph showing the proliferative capacity ofCD34⁺Thy-1⁺Lin⁻ cells purified from ABM and cultured individually in thepresence of 10% supernatant from COS cells transfected with TPO. Datawas determined as described in the legend to FIG. 1A.

[0022]FIG. 1C is a graph showing the proliferative capacity ofCD34⁺Thy-1^(+Lin) ⁻ cells purified from ABM and cultured individually inthe presence of 10% TPO COS supernatant and 10 ng/ml interleukin 3(IL-3). Data was determined as described in the legend to FIG. 1A.

[0023]FIG. 1D is a graph showing the proliferative capacity ofCD34⁺Thy-1^(+Lin) ⁻ cells purified from ABM and cultured individually inthe presence of 10% TPO COS supernatant and 25 ng/ml c-kit ligand (KL).Data was determined as described in the legend to FIG. 1A.

[0024]FIG. 1E is a graph showing the proliferative capacity ofCD34⁺Thy-1^(+Lin) ⁻ cells purified from ABM and cultured individually inthe presence of 10 ng/ml IL-3, 10 ng/ml IL-6, 10 ng/ml leukemiainhibitory factor (LIF), and 25 ng/ml KL. Data was determined asdescribed in the legend to FIG. 1A.

[0025] FIGS. 2A-2L are FACScan analysis of PKH26 and CD34 fluorescencein CD34⁺Lin⁻Rh123^(lo) cells. ABM CD34⁺Lin⁻Rh123^(lo) cells were labeledwith PKH26 membrane dye and cultured for 3 or 6 days without (control)or with TPO, KL, IL-3, IL-3+TPO, or KL+TPO in suspension cultures in theabsence of stroma. Following culture, the cells were reacted withanti-CD34-FITC antibody and PKH26 vs. CD34 fluorescence was analyzed ona FACScan analyzer. The percentages of CD34 and PKH26 positive ornegative cells are shown in the quadrants. Quadrants were set based onday 0 (uncultured) cells, and on cells cultured in the absence ofexogenous cytokines on day 3 and day 6. Day 3: FIG. 2A, control; FIG.2C, TPO; FIG. 2E, KL; FIG. 2G, IL-3; FIG. 2I, IL-3+TPO; FIG. 2K, KL+TPO.Day 6: FIG. 2B, control; FIG. 2D, TPO; FIG. 2F, KL; FIG. 2H, IL-3; FIG.2J, IL-3+TPO; FIG. 2L, KL+TPO.

[0026]FIG. 3A is a graph showing the effect of TPO on the expansion ofthe CD41b⁺ cell population of cultured CD34⁺Lin⁻ and CD34⁺Thy-1⁺Lin⁻cells.

[0027]FIG. 3B is a graph showing the effect of TPO on total cellsnumbers of cultured CD34⁺Lin⁻ and CD34⁺Thy-1⁺Lin⁻ cells.

DETAILED DESCRIPTION

[0028] Before the present invention and methods for using same aredescribed, it is to be understood that this invention is not limited tothe particular cell lines, mpl ligand, or methodology described. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting since the scope of the present invention will be limitedonly by the appended claims.

[0029] It must be noted that as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. Thus, forexample, reference to “a stem cell” includes a plurality of cells,including mixtures thereof, and reference to “a mpl ligand” includecompounds able to bind mpl with sufficient specificity to elicitmpl-mediated biological activity.

[0030] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although anymethodology and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdisclosing and describing the particular materials and methodologies forwhich the reference was cited in connection with.

DEFINITIONS

[0031] By the termn “mpl ligand” is meant a compound capable of bindingto mpl such that one or more mpl-mediated biological actions areinitiated. As herein disclosed, mpl-mediated biological activityincludes (1) promotion of the survival of stem cells in culture, suchthat the cell maintains the capability of self-renewal and the abilityto give rise to all hematopoietic cell lineages, (2) expansion of stemcell populations, such that the expanded cell population maintains thecapability of self-renewal and the ability to give rise to allhematopoietic cell lineages, and (3) activation of a quiescent stemcell, such that the stem cell is activated to divide and the resultingcells maintain the capability of self-renewal and the ability to giverise to all hematopoietic cell lineages. The mpl ligand in the inventioninitiates at least one mpl-mediated activity, and preferably two or morempl-mediated activities. Preferably, mpl ligand is thrombopoietin, andmore preferably, human thrombopoietin, and still more preferably,recombinant human thrombopoietin. The term “mpl ligand” also includesantibodies to the mpl receptor capable of binding to mpl such that oneor more of the above-described mpl-mediated biological actions areinitiated. Such antibodies may consist essentially of pooled monoclonalantibodies with different epitopic specificities, or be distinctmonoclonal antibodies. The term “mpl ligand” further includes mimeticmolecules, e.g., small molecules able to bind mpl such that one or moreof the above-described mpl-mediated biological actions are initiated.The methods and assays disclosed herein, combined with methods known tothe art, are utilized to construct libraries of mimetic molecules, andto screen the libraries such that a TPO mimetic is identified having therequisite biological activity.

[0032] By the term “stem cell” is meant hematopoietic cells which arecapable of self-regeneration when provided to a human subject in vivo,and may become lineage restricted progenitors, which furtherdifferentiate and expand into specific lineages. As used herein, “stemcells” refers to hematopoietic cells and not stem cells of other celltypes. Further, unless indicated otherwise, “stem cells” refers to humanhematopoietic stem cells.

[0033] The term “stem cell” or “pluripotent” stem cell are usedinterchangeably to mean a stem cell having (1) the ability to give riseto progeny in all defined hematopoietic lineages, and (2) stem cellscapable of fully reconstituting a seriously immunocompromised host inall blood cell types and their progeny, including the pluripotenthematopoietic stem cell, by self-renewal. A stem cell or pluripotentstem cell may be identified by expression of the cell surface markerCD34⁺.

[0034] Stem cells constitute only a small percentage of the total numberof hematopoietic cells. Hematopoietic cells are identifiable by thepresence of a variety of cell surface “markers.” Such markers may beeither specific to a particular lineage or progenitor cell or be presenton more than one cell type. CD34 is a marker found on stem cells and asignificant number of more differentiated progenitor cells. U.S. Pat.No. 4,714,680 describes a population of cells expressing the CD34marker.

[0035] As used herein, “stem cells” or “pluripotent stem cells” refersto a population of hematopoietic cells having all of the long-termengrafting potential in vivo. Animal models for long-term engraftingpotential of candidate human hematopoietic stem cell populations includethe SCID-hu bone model (Kyoizumi et al. (1992) Blood 79:1704; Murray etal. (1995) Blood 85:368-378) and the in utero sheep model (Zanjani etal. (1992) J. Clin. Invest. 89:1179). For a review of animal models ofhuman hematopoiesis, see Srour et al. (1992) J. Hematother. 1:143-153and the references cited therein. In vitro assays for stem cells includethe long-term culture-initiating cell (LTCIC) assay, based on a limitingdilution analysis of the number of clonogenic cells produced in astromal co-culture after 5-8 weeks (Sutherland et al. (1990) Proc. Natl.Acad. Sci. USA 87:3584-3588, and the cobblestone area forming cell(CAFC) assay, shown to correlate with the LTCIC assay and with long-termengrafting potential (Breems et al. (1994) Leukemia 8:1095).

[0036] For use in the present invention, a highly enriched stem cellpopulation is preferred. An example of an enriched stem cell populationis a population of cells selected by expression of the CD34 marker. InLTCIC assays, a population enriched in CD34⁺ cells will typically havean LTCIC frequency in the range of 1/50 to 1/500, more usually in therange of 1/50 to 1/200. Preferably, the stem cell population will bemore highly enriched for stem cells than that provided by a populationselected on the basis of CD34⁺ expression alone. By use of varioustechniques described more fully below, a highly enriched stem cellpopulation may be obtained. A highly enriched stem cell population willtypically have an LTCIC frequency in the range of 1/5 to 1/100, moreusually in the range of 1/10 to 1/50. Preferably, it will have an LTCICfrequency of at least 1/50. Exemplary of a highly enriched stem cellpopulation is a population having the CD34⁺Lin⁻ or CD34⁺Thy-1⁺Lin⁻pheotype as described in U.S. Pat. No. 5,061,620, incorporated herein byreference to disclose and describe such cells. A population of thisphenotype will typically have an average LTCIC frequency ofapproximately 1/20 (Murray et al. (1995) supra; Lansdorp et al. (1993)J. Exp. Med. 177:1331). LTCIC frequencies are known to correlate withCAFC frequencies (Reading et al. (1994) supra).

[0037] Most preferably, the stem cell population will be enriched forthe ability to efflux the mitochondrial dye rhodamine123 (Rh123). Theability to efflux Rh123 is a characteristic associated with the mostprimitive pluripotent human stem cell (Udomsakdi et al. (1991) Exp.Hematol. 19:338; Srour et al. (1991) Cytometry 12:179; Chaudhary &Roninson (1991) Cell 66:85). Thus, in the most preferred embodiment, thestem cell population is characterized by having the CD34⁺Lin⁻Rho123^(lo)or CD34⁺Thy-1⁺Lin⁻Rho123^(lo) phenotype. It will be appreciated by thoseof skill in the art that the enrichment provided in any stem cellpopulation will be dependent both on the selection criteria used, aswell as the purity achieved by the given selection techniques. Further,the LTCIC or CAFC frequencies obtained will vary depending on the assayconditions such as the stromal cells and cytokines used, although theenrichment in stem cell activity over that present in whole bone marrowwill be comparable.

[0038] Stem cells may be isolated from any known human source of stemcells, including bone marrow, both adult and fetal, mobilized peripheralblood (MPB) and umbilical cord blood. Initially, bone marrow cells maybe obtained from a source of bone marrow, including ilium (e.g. from thehip bone via the iliac crest), tibia, femora, spine, or other bonecavities. Other sources of stem cells include embryonic yolk sac, fetalliver, and fetal spleen.

[0039] For isolation of bone marrow, an appropriate solution may be usedto flush the bone, including saline solution, conveniently supplementedwith fetal calf serum (FCS) or other naturally occurring factors, inconjunction with an acceptable buffer at low concentration, generallyfrom about 5-25 mM. Convenient buffers include HEPES, phosphate buffersand lactate buffers. Otherwise bone marrow may be aspirated from thebone in accordance with conventional techniques well known to thoseskilled in the art.

[0040] Methods for mobilizing stem cells into the peripheral blood areknown in the art and generally involve treatment with chemotherapeuticdrugs, e.g, cytoxan, cyclophosphamide, VP-16, and cytokines such asGM-CSF, G-CSF, or IL-3, or combinations thereof. Typically, apheresisfor total white cells begins when the total white cell count reaches500-2000 cells/μl and the platelet count reaches 50,000/μl. Dailyleukapheris samples may be monitored for the presence of CD34⁺ and/orThy-1⁺ cells to determine the peak of stem cell mobilization and, hence,the optimal time for harvesting peripheral blood stem cells.

[0041] Various techniques may be employed to separate the cells byinitially removing cells of dedicated lineage (“lineage-committed”cells). Monoclonal antibodies are particularly useful for identifyingmarkers associated with particular cell lineages and/or stages ofdifferentiation. The antibodies may be attached to a solid support toallow for crude separation. The separation techniques employed shouldmaximize the viability of the fraction to be collected.

[0042] The use of separation techniques include those based ondifferences in physical (density gradient centrifugation andcounter-flow centrifugal elutriation), cell surface (lectin and antibodyaffinity), and vital staining properties (mitochondria-binding dyerhodamine 123 and DNA-binding dye Hoechst 33342). Procedures forseparation may include magnetic separation, using antibody-coatedmagnetic beads, affinity chromatography, cytotoxic agents joined to amonoclonal antibody or used in conjunction with a monoclonal antibody,including complement and cytotoxins, and “panning” with antibodyattached to a solid matrix or any other convenient technique. Techniquesproviding accurate separation include flow cytometry which can havevarying degrees of sophistication, e.g., a plurality of color channels,low angle and obtuse light scattering detecting channels, impedancechannels, etc.

[0043] A large proportion of the differentiated cells may be removed byinitially using a relatively crude separation, where major cellpopulation lineages of the hematopoietic system, such as lymphocytic andmyelomonocytic, are removed, as well as lymphocytic populations, such asmegakaryocytic, mast cells, eosinophils and basophils. Usually, at leastabout 70 to 90 percent of the hematopoietic cells will be removed.

[0044] Concomitantly or subsequent to a gross separation providing forpositive selection, e.g. using the CD34 marker, a negative selection maybe carried out, where antibodies to lineage-specific markers present ondedicated cells are employed. For the most part, these markers includeCD2⁻, CD3⁻, CD7⁻, CD8⁻, CD10⁻, CD14⁻, CD15⁻CD16⁻, CD19⁻, CD20⁻, CD33⁻,CD38⁻, CD71⁻, HLA-DR³¹ , and glycophorin A; preferably including atleast CD2⁻, CD14⁻, CD15⁻, CD16⁻, CD19⁻ and glycophorin A; and normallyincluding at least CD14⁻ and CD15⁻. As used herein, Lin⁻ refers to acell population lacking at least one lineage specific marker. Thehematopoietic cell composition substantially depleted of dedicated cellsmay be further separated using selection for Thy-1⁺ and/or Rho123^(lo),whereby a highly enriched stem cell population is achieved.

[0045] The purified stem cells have low side scatter and low to mediumforward scatter profiles by FACS analysis. Cytospin preparations showthe enriched stem cells to have a size between mature lymphoid cells andmature granulocytes. Cells may be selected based on light-scatterproperties as well as their expression of various cell surface antigens.

[0046] Preferably, cells are initially separated by a coarse separation,followed by a fine separation, with positive selection of a markerassociated with stem cells and negative selection for markers associatedwith lineage committed cells. Compositions highly enriched in stem cellsmay be achieved in this manner. The desired stem cells are exemplifiedby a population with the CD34⁺Thy-1⁺Lin⁻ phenotype, and arecharacterized by being able to be maintained in culture for extendedperiods of time, being capable of selection and transfer to secondaryand higher order cultures, and being capable of differentiating into thevarious lymphocytic and myelomonocytic lineages, particularly B- andT-lymphocytes, monocytes, macrophages, neutrophils, erythrocytes and thelike.

[0047] The stem cells may be grown in culture in an appropriate nutrientmedium, including conditioned medium, a co-culture with an appropriatestromal cell line, or a medium comprising a combination of growthfactors sufficient to maintain the growth of hematopoietic cells. Forconditioned media or co-cultures, various stromal cell lines may beused. Since human stromal cell lines are not required, other stromalcell lines may be employed, including rodentiae, particularly murine.Suitable murine stromal cell lines include AC3 and AC6, which aredescribed in Whitlock et al. (1987) Cell 48:1009-1021. Preferably, thestromal cell line used is a passage of AC6, AC6.21 (otherwise referredto as SyS1).

[0048] The compositions comprising stem cells can be tested for theability to produce myeloid cells and lymphoid cells in appropriatestromal cell co-cultures. The stromal cells may come from varioussources, including human, porcine or murine, by selection for theability to maintain stem cells, and the like. Preferably, the stromalcells are AC3 or AC6, most preferably AC6.21, and the ability to produceB lymphocytes and myeloid cells is determined in cultures supplied withLIF and IL-6. The stem cells also give rise to B-cells, T-cells andmyelomonocytic cells in the in vivo assays described below.

[0049] To demonstrate differentiation to T-cells, fetal thymus isisolated and cultured from 4-7 days at about 25° C., so as tosubstantially deplete the lymphoid population. The cells to be testedfor T-cell activity are then microinjected into the thymus tissue, wherethe HLA of the population which is injected is mismatched with the HLAof the thymus cells. The thymus tissue may then be transplanted into ascid/scid mouse as described in U.S. Pat. No. 5,147,784, hereinspecifically incorporated by reference, particularly transplanting underthe kidney capsule. After 6 weeks, the thymus tissue is harvested andanalyzed for donor-derived CD4⁺ and/or CD8⁺ T-cells by flow cytometry.

[0050] Further demonstration of the long-term repopulating ability ofthe stem cell populations useful in the invention may be accomplished bythe detection of continued myeloid and B-lymphoid cell production in theSCID-hu bone model (Kyoizumi et al. (1992) Blood 79:1704). To analyzethis, one may isolate human fetal bone and transfer a longitudinallysliced portion of this bone into the mammary fat pad of a scid/scidanimal. The bone cavity is diminished in endogenous cells by whole bodyirradiation of the mouse host prior to injection of the test donorpopulation. The HLA of the population which is injected is mismatchedwith the HLA of the recipient bone cells. Stem cells from humanhematopoietic sources sustain B lymphopoiesis and myelopoiesis in theSCID-hu bone model.

[0051] For RBCs, one may use conventional techniques to identify burstforming unit-erythroid (BFU-E) units, for example methylcelluloseculture, demonstrating that the cells are capable of developing into theerythroid lineage (Metcalf (1977) In: Recent Results in Cancer Research61. Springer-Verlag, Berlin, pp. 1-227).

Effect of Thrombovoietin on Stem Cells

[0052] The human mpl transcript in highly purified bone marrow andmobilized peripheral blood CD34⁺Thy-1⁺Lin⁻ stem cells was demonstratedby RT-PCR (Example 3). However, it was not know if the receptor isexpressed on the cell surface or if a receptor ligand would have anyeffect on the cells.

[0053] The present study was initiated to determine whether TPO has anyeffect on primitive human hematopoietic stem cells (“HSC”), and if so,to define its range of activities. Initially, human TPO was cloned andsequenced based on the published sequence as described in Example 1.

[0054] Single CD34⁺Thy-1⁺Lin⁻ cells from adult bone marrow (ABM) werecultured individually in Terasaki wells on the murine stromal cellmonolayer, SyS1, with TPO alone or in combination with cytokines IL-3,KL, IL-6, or LIF (Example 4). The growth of each cell was tracked byimage acquisition, as described in Example 4. FIGS. 1A-1E show themaximum proliferation achieved over a 4 week period by eachCD34⁺Thy-1⁺Lin⁻ cell plated under various cytokine conditions from arepresentative ABM tissue. TPO alone supported a plating efficiency of74% and an average cell production of 454 cells per cell. Platingefficiency is defined as the number of cells that divided one or moretimes/number of cells plated. When IL-3 was added, cell production wasenhanced approximately 4-fold. This level of proliferation was similarto that achieved in the presence of combined IL-3, IL-6, LIF and KL. Theresults show that TPO acts directly on human CD34⁺Thy-1⁺Lin⁻ cells toinduce cycling of primitive noncommitted human hematopoietic progenitors(e.g., expressing CD34 but not lineage antigens), and thatco-stimulation with IL-3 increases total cell production.

[0055] Approximately 50% of single CD34⁺Thy-1⁺Lin⁻ cells grown in thepresence of TPO displayed MK differentiation, as measured by size andCD41b antigen expression (Example 4). CD41b antigen expression ischaracteristic of cells committed to the MK lineage. These resultsdemonstrate that TPO supports stem cell differentiation into MK lineage.

[0056] To quantitatively demonstrate that TPO was stimulating singlestem cells to produce multiple MK progenitors prior to maturation, thenumber of CFU-MK present in populations of day 0 ABM CD34⁺Thy-1⁺Lin⁻cells were compared to the number obtained from wells of proliferatingblast cells that grew from a single CD34⁺Thy-1⁺Lin⁻ cell in the presenceof TPO. In one representative experiment, day 0 CD34⁺Thy-1⁺Lin⁻ cellsproduced 0.0026 CFU-MK per cell, compared to blast cell populationsexpanded from a single CD34⁺Thy-1⁺Lin⁻ cell, which produced an averageof 8.3 CFU-MK per original cell (range 1-18) in the presence of TPO andIL-3. This represents an amplification in MK progenitor productionduring culture of 3,200-fold, and indicates that TPO can act on singlehuman stem cells and permit commitment to the MK lineage, MK progenitorexpansion, and MK maturation in culture. - In addition to promoting MKproliferation and differentiation, it has now been found that TPO actson primitive HSC to promote expansion without differentiation into theMK lineage. This finding is surprising in light of the report that onlythe MK lineage is defective in mpl-deficient transgenic mice (Guerney etal. (1994) Science 265:1445). Approximately 50% of the singleCD34⁺Thy-1⁺Lin⁻ cells from ABM that grew in the presence of TPO did notdisplay MK differentiation. A small proportion (10% of cells that grew)developed directly into large granular cells with macrophagecharacteristics. The remaining 40% of cells that grew formed tightlyapposed clusters of blast cells (cobblestone areas) which did notdifferentiate to large refractile MK or large granular macrophage cellsover a four week period. The addition of IL-3 to TPO increased thefrequency of cells exhibiting a non-megakaryocytic blast cell expansionpattern from 40% to approximately 80% of single cells that grew, andincreased the proliferation of single cells approximately 4-fold suchthat some wells reached the maximum capacity of the Terasaki well(approximately 10,000 cells).

[0057] To determine whether other cytokines alone, or in combinationwith TPO could support proliferation of primitive CD34⁺ cells, cytokineswith known activities on early hematopoietic cells were tested. AlthoughCD34⁺ cells were detected in cell populations expanded usingIL-6+LIF+IL-3+KL, the highest percentages of CD34⁺ cells were alwaysfound in wells which contained TPO (Table 1). A typical TPO-expandedblast population of approximately 1,000 cells, of which 2% are CD34positive, represents an approximate 20-fold expansion of CD34⁺ (Table1).

[0058] A stromal support layer (SyS1) was used in the long-term cultureassays herein described because previous observations showed that singleor small numbers of HSC cultured under stroma-free conditions displayedmore rapid differentiation and lower total cell production than thosecultured on a stromal layer. SyS1 culture supernatants alone cannotsubstitute for the stromal cells for support of hematopoieticprogenitors. In addition, single HSC plated on SyS1 without exogenoushuman cytokine addition show an extremely low growth frequency. Theabove findings made with single HSC grown on a stromal layer weresubstantiated by showing that in bulk cultures grown in the absence ofstroma, purified recombinant TPO alone can activate quiescent HSC.

[0059] PKH26 is a fluorescent dye which labels cell membranes andreduces in intensity with each cell division. The effect of TPO on PKH26fluorescence relative to CD34 expression was analyzed in stromal-freebulk cultures. For these experiments, an enriched quiescent(CD34⁺Lin⁻Rh123^(lo)) stem cell population was tested. PKH26 dye markingof cell membranes was used to obtain quantitation of cell divisions inconjunction with expression of the CD34 antigen to determine the effectsof various cytokine combinations on stem cell proliferation anddifferentiation. TPO alone was able to stimulate these quiescent HSC tobegin cycling within 3 days (FIG. 2C), and still retain high levels ofCD34 expression after 6 days of culture (FIG. 2D). TPQ alone causedgreater activation than KL alone at 3 (FIG. 2E) and 6 days (FIG. 2F).When TPO and KL were combined, they acted synergistically to increasecell cycling by day 6 (FIG. 2L). IL-3 plus TPO produced much more cellproliferation than TPO alone, but caused more differentiation than TPOalone or TPO+KL (e.g., loss of CD34 expression) (FIGS. 2I and 2J).

Therapeutic Use of ThromboRoietin

[0060] The present invention is based on the discovery that mpl ligand,thrombopoietin, has unique and unexpected effects on human stem cells.As established by the evidence provided herein, exposure of humanlong-term repopulating stem cells to TPO results in cell expansion withminimal differentiation. TPO also causes expansion of human stem cellswith subsequent differentiation to the MK lineage, as well as non-MKcells. Further, TPO is shown to activate quiescent human stem cells intocycle more quickly than other known cytokines. Additionally, TPO isherein shown to promote the survival of human stem cells in in vitroculture.

[0061] The subset of primitive human hematopoietic cells shown herein torespond to TPO are long-term repopulating or pluripotent stem cells,characterized by the ability to give rise to cells which retain thecapability of self-renewal, and to proliferate and differentiate -intocells of all hematopoietic lineages.

[0062] Use of TPO for Expansion of Long-Term Repopulating CellPopulations. The ability of TPO to induce extensive proliferation of aprimitive human stem cell subpopulation without loss of the pluripotentcapacity has important clinical implications for restoration ofhematopoietic capability in subjects in which hematopoietic capabilityis lost or threatened. Accordingly, the invention features the use ofTPO for in vitro expansion of a human long-term repopulating cellpopulation. This is particularly useful for re-establishinghematopoietic capability in patients in which native hematopoieticcapability has been partially, substantially, or completely compromised.Stem cells from any tissue are removed from a human subject, expanded invitro by exposure to TPO, and the expanded cells are returned to thepatient. If necessary, the process may be repeated to ensure substantialrepopulation of the stem cells. The expanded stem cell populationreturned to the subject retain pluripotent characteristics, e.g.,self-renewal and ability to generate cells of all hematopoieticlineages. By combination of various cytokines the expanded cellpopulation will include progenitor cells and more mature cells of thevarious hematopoietic lineages (e.g., megakaryocytes, neutrophils) inaddition to stem cells to provide a cell population that will provideboth short-term and long-term repopulation potential.

[0063] Stems cells are preferably isolated from bone marrow or mobilizedperipheral blood, and more preferentially from bone marrow. Expansionprocedures may be conducted either with or without stromal cells.Stromal cells may be freshly isolated from bone marrow or from clonedstromal cell lines. Such lines may be human, murine, or porcine;preferably, the cell line is AC6.21, as described in the Examples below.For clinical applications, it is preferred to culture the stem cells inthe absence of stromal cells. During expansion, TPO may be present onlyduring the initial course of the stem cell growth and expansion, usuallyat least 24 hours, more usually at least about 48 hours to 4 days, ormore preferably is maintained during the course of the expansion. Duringcell expansion, TPO is present in a concentration range of 10-200 ng/ml;more preferably, TPO is present in a concentration range of about 10-100ng/ml. In addition, TPO may be combined with the use of other growthfactor and cytokines, such as IL-3. During stem cell expansion, TPO ispresent in a concentration range of between about 1 ng/ml to about 200ng/ml, preferably in the range of between about 50 ng/ml and 100 ng/ml,most preferably about 50 ng/ml. When cell expansion is conducted in thepresence of TPO and IL-3, IL-3 is present in a concentration range ofbetween about 1 ng/ml to about 100 ng/ml, preferably in the range ofbetween about 5 ng/ml and 25 ng/ml, most preferably about 10 ng/ml.Additional regulatory factors may be present, for example, Stl (50 -100ng/ml), LIF (50 ng/ml), IL-6 (2-100 ng/ml), MIP-1α (2-100 ng/ml),Flk2/Flt3 (2-100 ng/ml), G-CSF (2-20 ng/ml), GM-CSF (2-20 ng/ml), IL-1(2-100 ng/ml), and IL-11 (2-100 ng/ml). Various in vitro and in vivotests known to the art may be employed to ensure that the pluripotentcapability of the stem cells has been maintained.

[0064] Use of Thrombopoietin in Gene Therapy. TPO has been shown hereinto stimulate a quiescent human long-term repopulating stem cellpopulation to begin actively dividing without differentiation, e.g.,culturing long-term repopulating stem cells in the presence of TPOresults in the generation of cells which retain the capability ofself-renewal and ability to give rise to cells of all hematopoieticlineages. This discovery is particularly important for transduction ofhuman stem cells with exogenous genes because retroviral vectors requiretarget cells to be cycling for stable integration of the retroviral DNA.The ability to modify a human pluripotent cell population should providelong-term repopulation of an individual with the modified cells andtheir progeny, which will express the desired gene product. By contrast,gene transfer into more mature hematopoietic cells, such as T cells, atbest, provides only transient therapeutic benefit. Thus, the use of TPOfor transduction of stem cells satisfies the current world-wide effortto find effective methods of genetically modifying stem cells. Forreviews of genetic modification of stem cells see Brenner (1993) J.Hematother. 2:7-17; Miller (1992) Nature 357:455-460; and Nienhuis(1991) Cancer 67:2700-2704.

[0065] While retroviral vectors may be used to genetically modify apopulation of human long-term repopulating stem cells, other methods maybe used, such as liposome-mediated gene transfer or adeno-associatedviral vectors. Retroviral vectors have been the primary vehicle due tothe generally high rate of gene transfer obtained in experiments withcell lines, and the ability to obtain stable integration of the geneticmaterial, which ensures that the progeny of the modified cell willcontain the transferred genetic material. Retroviral vectors and theiruse in the transfer and expression of foreign genes are reviewed inGilboa (1988) Adv. Exp. Med. Biol. 241:29; Luskey et al. (1990) Ann.N.Y. Acad. Sci. 612:398; and Smith (1992) J. Hematother. 1:155-166.

[0066] Hematopoietic stem cells are removed from a human patient, and apopulation of long-term repopulating stem cells isolated. These cellsmay be optionally expanded prior to or after modification bytransduction with a vector carrying the desired gene. The modified cellsare then restored to the human patient for expression of the foreigngene. The patient may be treated to partially, substantially, orcompletely ablate the native hematopoietic capability prior torestoration of the modified stem cells. Preferably, after completion ofthe treatment of the host, the modified stem cells may then be restoredto the host to provide for expression of the foreign gene. The methodsof stem cell removal, host ablation and stem cell repopulation are knownin the art. If necessary, the process may be repeated to ensuresubstantial repopulation of the modified stem cells.

[0067] During optional expansion, TPO alone or combined with othergrowth factors may be present only during the initial course of the stemcell growth and expansion, usually at least 24 hours, more usually atleast about 48 hours to 4 days, or may be maintained during the courseof the expansion. Example 8 below describes a protocol for stem celltransduction in the presence of TPO. For use in clinical settings, it ispreferable to transduce the stem cells without prior or subsequentexpansion.

[0068] Transduction may be accomplished by the direct co-culture of stemcells with producer cells, e.g. by the method of Bregni et al. (1992)Blood 80:1418-1422. For clinical applications, however, transduction byculturing the stem cells with viral supernatant alone or with purifiedviral preparations, in the absence of stromal cells, is preferred.Transductions may be performed by culturing the stem cells with thevirus for from about four hours to six days. Preferably, transduction iscarried out for three days, with the media replaced daily with mediacontaining fresh retrovirus. Alternatively, the stem cells may becultured in the presence of virus for several hours, e.g., four hours,daily for three to four days, with fresh media replacing thevirus-containing media each day. Polycations, such as protamine sulfate,polybrene and the like, will generally be included to promote binding.Protamine sulfate and polybrene are typically used in the range of 4μg/ml.

[0069] Other cytokines may also be added, including, e.g., IL-3, IL-6,LIF, steel factor (Stl) GM-CSF, G-CSF, MIP-1α, and Flk2/Flt3, preferablyincluding Stl. The factors employed may be naturally occurring orsynthetic, e.g., prepared recombinantly, and preferably human. Theamount of the factors will generally be in the range of about 1 ng/ml to200 ng/ml. Generally, for TPO, the concentration will be in the range ofabout 1 ng/ml to 200 ng/ml, more usually 5 ng/ml to 100 ng/ml, andoptimally about 10 ng/ml to 50 ng/ml; for Stl, the concentration rangewill be in the range of 10 ng/ml to 200 ng/ml, and more usually 50 ng/mlto 100 ng/ml; for LIF, the concentration will be in the range of about 1ng/ml to 100 ng/ml, more usually 10 ng/ml to 80 ng/ml, and optimallyabout 50 ng/ml; for IL-3, the concentration will be in the range ofabout 5 ng/ml to 100 ng/ml, more usually 5 ng/ml to 50 ng/ml; for IL-6,the concentration will be in the range of about 5 ng/ml to 50 ng/ml,more usually 5 ng/ml to 20 ng/m, and for GM-CSF, the concentration willgenerally be 5 ng/ml to 50 ng/ml, more usually 5 ng/ml to 20 ng/ml.

[0070] To ensure that the stem cells have been successfully modified,PCR may be used to amplify vector specific sequences in the transducedstem cells or their progeny. In addition, the cells may be grown undervarious conditions to ensure that they are capable of maturation to allof the hematopoietic lineages while maintaining the capability, asappropriate, of the introduced DNA. Various in vitro and in vivo testsdescribed above may be employed to ensure that the pluripotentcapability of the stem cells has been maintained.

[0071] Gene Therapy Applications. Gene transfer into stem cells may beused to treat a variety of neoplastic, infectious or genetic diseases.For example, one may introduce genes that confer resistance tochemotherapeutic agents, thereby protecting the progeny hematopoieticcells, allowing higher doses of chemotherapy and thereby improving thetherapeutic benefit of treatment. For instance, the mdr1 gene may beintroduced into stem cells to provide increased resistance to a widevariety of drugs including taxol, which are exported by the mdr1 geneproduct, in combination with the administration of chemotherapeuticssuch as taxol, e.g. for breast cancer treatment. Similarly, genes thatprovide increased resistance to alkylating agents, such as melphalan,may be introduced into stem cells in conjunction with high dosechemotherapy.

[0072] For viral infections that primarily affect hematolymphoid cells,stem cells may be modified to endow the progeny with resistance to theinfectious agent. In the case of human immnunodeficiency virus (HIV),for example, specific antisense or ribozyme sequences may be introducedthat interfere with viral infection or replication in the target cells.Alternatively, the introduced gene products may serve as “decoys” bybinding essential viral proteins, thereby interfering with the normalviral life cycle and inhibiting replication.

[0073] Alternatively, stem cells may be modified to produce a product tocorrect a genetic deficiency, or where the host has acquired a geneticdeficiency through a subsequent disease. Genes that may correct agenetic deficiency include adenosine deaminase for the treatment of ADAsevere combined immunodeficiency; glucocerebrosidase for the treatmentof Gaucher's disease; β-globin for the treatment of sickle cell anemia;and Factor VIII or Factor IX for the treatment of hemophilia; tumorantigen-specific T-cell receptors for immunotherapy of cancer, andcytokines for treatment of cancer and cytokine-related disorders.

[0074] Expression of the transferred gene can be controlled in a varietyof ways depending on the purpose of gene transfer and the desiredeffect. Thus, the introduced gene may be put under the control of apromoter that will cause the gene to be expressed constitutively, onlyunder specific physiologic conditions, or in particular cell types.Examples of promoters that may be used to cause expression of theintroduced sequence in specific cell types include Granzyme A andGranzyme B for expression in T-cells and NK cells, the CD34 promoter forexpression in stem and progenitor cells, the CD8 promoter for expressionin cytotoxic T-cells, and the CD11b promoter for expression in myeloidcells. Inducible promoters may be used for gene expression under certainphysiologic conditions. The therapeutic benefit may be further increasedby targeting the gene product to the appropriate cellular location, forexample, the nucleus, by attaching the appropriate localizing sequences.In addition, by appropriate use of inducible promoters, expression ofvarious protein products can be achieved in response to particularstimuli such as chemicals, chemo-attractants, particular ligands, andthe like.

EXAMPLE 1 Cloning and Expression of Thrombovoietin

[0075] TPO cDNA was cloned using the polymerase chain reaction fromhuman fetal liver cDNA (Clontech, Palo Alto, Calif.), usingoligonucleotides based on the published sequence (de Sauvage et al.(1994) supra), and expressed in COS cells. TPO-containing COSsupernatants were shown to be active in stimulating the proliferation ofmpl-expressing BaF3 cells (activity was 3×105 U/ml, where 50 U/ml=50%maximum activity), which were constructed by stable transfection of BaF3cells with a human mpl expression construct as previously described (deSauvage et al. (1994) supra). Mock transfected COS supernatant (COScontrol) was harvested from COS cells transfected with an emptyexpression vector, and had no effect on the proliferation of BaF3 cells.

EXAMPLE 2. Cell Selection

[0076] Enrichment of progenitor cells from human adult bone marrow andmobilized peripheral blood. Human adult bone marrow (ABM) CD34+ cellswere enriched as follows. Briefly, fresh 20 ml bone marrow aspiratesfrom normal volunteer donors were obtained from Stanford UniversityMedical Center (Palo Alto, Calif.) or Scripps Research Institute (LaJolla, Calif.). Mononuclear cells (density <1.077) were isolated from aFicoll (Pharmacia, Milwaukee, Wis.) density gradient in cell separationmedium [RPMI 1640 (JRH Biosciences, Lenexa, Kans.), 10% fetal bovineserum (FBS) (Gemini Bioproducts, Calabasas, Calif.), 50 U/ml penicillinand 50 μg/ml streptomycin (JRH Biosciences)]. CD34 positive selectionwas performed by the method of Sutherland et. al (1992) Exp. Hematol.20:590). The glycoprotease from Pasteurella haemolytica used for thebead release step in this procedure has been shown not to effectsubsequent ex vivo expansion of progenitors (Marsh et al. (1992)Leukemia 6:929).

[0077] Stem cells were mobilized into the peripheral blood (MPB) ofmultiple myeloma patients and collected by leukapheresis as described byMurray et al. (1995) supra. MPB CD34⁺ cells were enriched fromleukapheresis products by using a procedure involving elutriation,phenylmethylester lysis of granulocytes, and ammonium chloride lysis ofred blood cells and CD34 positive selection as described by Sutherlandet al. (1992) supra.

[0078] Antibodies. TuK3 (anti-CD34) directly conjugated tosulpharhodamine (SR) and GM201 (anti-human Thy-1) directly conjugated tophycoerythrin (PE) were used to purify primitive progenitors by flowcytometry. FLOPC 21 mouse IgG3 (Sigma, St. Louis, Mo.) and purifiedmouse IgG1 (Becton Dickinson, Mountain View, Calif.) were conjugated toSR and PE, respectively, and used as controls for CD34 and Thy-1staining. Fluorescein isothiocyanate (FITC)-conjugated lineageantibodies were used to exclude lineage positive cells from selection[Leu-5b (anti-CD2), Leu-M3 (anti-CD14), Leu-M1 (anti-CD15), Leu-11a(anti-CD16), Leu-12 (anti-CD19)], FlTC-conjugated mouse IgG1 and IgG2a,PE and FlTC-conjugated HPCA2 (anti-CD34) and mouse IgG1 were purchasedfrom Becton Dickinson. FlTC-conjugated antibody D2.10 (anti-glycophorin)was purchased from AMAC (Westbrook, Me.). FlTC-conjugated mouse IgM waspurchased from Sigma (St. Louis Mo.). Human gamma globulin (Gamimune),was purchased from Miles Inc. (Elkhart, Ind.).

[0079] Fluorescent labeling and flow cytometry. CD34⁺ enrichedhematopoietic cells were adjusted to a concentration of 2×10⁶/ml instaining medium [RPMI 1640 without phenol red, 2% FBS, and 10 mM HEPES(Sigma, St. Louis, Mo.)] and labeled using the following procedureperformed on ice.

[0080] Heat inactivated human gamma globulin (Gamimune) was added to thecell suspension at a concentration of 1 mg/ml to block Fc receptorbinding sites. Cells were incubated with anti-CD34-SR (10 μg/ml),anti-Thy-1-PE (10 μg/ml), and a mixture of FlTC-conjugated antibodiesdirected against a panel of lineage markers. The mixture includedanti-CD2, anti-CD14, anti-CD15, anti-CD16, anti-CD19, andanti-glycophorin A. Irrelevant mouse IgG3-SR (10 μg/ml) and IgG1-PE (10μg/ml) antibodies were used as controls for CD34 and Thy-1 staining.FlTC-conjugated mouse IgG1, IgG2a, and IgM were used as controls for thelineage panel. Incubation time for staining was 20 minutes followed by athree ml wash with staining medium.

[0081] Following antibody staining, cells were resuspended at aconcentration of 10⁶ cells/ml in staining medium containing 1 μg/mlpropidium iodide (Pl) (Molecular Probes Inc., Eugene Oreg.).Fluorescently stained cells were analyzed and sorted on a FACStar Pluscell sorter (Becton Dickinson). Dead cells (Pl positive) and lineagepositive cells (stained with an intensity above the isotype matchedcontrol antibody staining) were excluded from the sort gate. TheCD34⁺Thy-1⁺ and, in some cases, CD34⁺Thy-1⁻, fractions were sorted fromthe live, lineage-negative cells (CD34⁺Thy-1⁺Lin⁻ or CD34⁺Thy-1-Lin⁻).

[0082] CD34⁺ selected ABM cells were loaded with rhodamine 123 dye(Rh123) (Molecular Probes, Eugene, Oreg.), and allowed to efflux at 37°C. for 20 min, according to the method of Spangrude et al. (1988)Science 241:58-62, herein specifically incorporated by reference. Thecells were then chilled to 4° C. and reacted with anti-CD34-SR and thesame lineage marker antibodies as described above except conjugated toPE. Cells with a CD34⁺Lin⁻Rh123^(lo)phenotype were purified by flowcytometry.

EXAMPLE 3 PCR Detection of MPL in Bone Marrow and Mobilized PeripheralBlood Samples.

[0083] Cell lysates were prepared from total human bone marrow cells andbone marrow or mobilized peripheral blood cells of the phenotypesCD34⁺Thy-1⁺Lin⁻ (Thy⁺) and CD34⁺Thy-1⁻Lin ⁻ (Thy⁻), isolated asdescribed in Example 2. RNA was prepared using RNA STAT60 (Tel-Test,Inc.) according to the manufacturer's instructions. CDNA was made fromeach of the RNA samples using BRL Superscript RT following themanufacturer's instructions. One-fifth of the cDNA was then used for thefirst degenerate PCR reaction. The degenerate PCR primers designed toamplify sequences related to the Epo receptor and mpl receptor of theclass I hemopoietin receptor family are as follows: Sense primer HEpM1-5(SEQ ID NO:1) 5′-GCTATTGCGGCCGCGAATTCGGARGAYYTIINITGYTTYTGG-3′:Antisense primer HEpM2-3 (SEQ ID NO:2)5′-GCTATTCTCGAGATCGATSWCCAITCRCTCCAIIINCC-3′: Antisense primer HEpM3-3(SEQ ID NO:3) 5′-GCTATTCTCGAGATCGATSWCCAINIRCTCCAIIINCC-3′:

[0084] (R=A,G; Y=C,T, I=inosine; S=G,C; W=A,T). Restriction sites wereadded at the 5′ end of each primer. One set of the reaction wasperformed with HEpM1-5 and HEpM2-3, and another reaction with wasperformed with HEpM1-5 and HEpM3-3. In each case, a smear was obtained.

[0085] The two PCR products (2 μl of each) were mixed for the second PCRreaction. The second PCR was carried out using specific primers to mpl.The following two sets of primers were used: Set A: sense primermp1-5′ (317-336) (SEQ ID NO:4) 5′-CGCTGCACCTCTGGGTGAAG-3′: antisenseprimer mp1-3′ (568-588) (SEQ ID NO:5) 5′-AGCAGGGCAGCAGGTTTCTGT-3′: SetB: sense primer mp1-5′ (317-336) (SEQ ID NO:4) and antisense primermp1-3-HI (754-774) (SEQ ID NO:6) 5′-GCTGCGCAGCTGCAGCCAGTA-3′:

[0086] Results. In total bone marrow samples, mpl was detected withmpl-5′/mpl-3′ primers alone. In sorted bone marrow samples, mpl wasdetected in both Thy⁺ and Thy⁻ samples using both sets of primers. mplwas detected using both sets of primers in peripheral blood samples(Thy⁺ and Thy⁻).

EXAMPLE 4 Effect of TPO on Single Cell Cultures of Stem Cells.

[0087] Terasaki plates used for single cell deposition were preseededwith a murine stromal cell line (AC6.21, also referred to as SyS1)described previously (Baum et al. (1992) Proc. Natl. Acad. Sci. USA89:2804; DiGiusto et al. (1994) Blood 84:421; Murray et al. (1995)supra), at approximately 50 cells per well in 20 μl of stromal cellculture medium [RPMI 1640, 5% FBS, 10⁵ M 2-mercaptoethanol (2-ME)(Sigma), 4 mM glutamine (JRH Biosciences), 50 U/ml penicillin, and 50μg/ml streptomycin]. Stromal cells were allowed to form a confluentmonolayer by culturing for 4-7 days. At confluence monolayers wereirradiated (1,000 rads from a cesium source) to prevent overgrowth.Immediately prior to seeding of hematopoietic cells, 10 μl of medium wasremoved and replaced with 10 μl stem cell culture medium (SCCM) [IMDM(JRH Biosciences), 5% pooled human plasma (HP), 7.5×10⁻³ M α-TG (AldrichChemical Co. Milwaukee, Wis.), 10⁻⁵ M 2-ME, 100 mM Na pyruvate (JRHBiosciences), 4 mM glutamine, 50 U/ml penicillin, 50 μg/mlstreptomycin]. The culture medium was chosen for maintenance of MKintegrity which included human plasma (HP) and α-TG. SCCM wassupplemented with combinations of cytokines as indicated. Cytokinesincluded recombinant human (rh)IL-3 (10 ng/ml) (Genzyme, Cambridge,Mass.), rhIL-6 (10 ng/ml) (Sandoz, Basel, Switzerland), rhKL (25 ng/ml)and rhLIF (10 ng/ml) (Tago/Biosource, Camarillo, Calif.) and 10%supernatant collected from COS cells transfected with a TPO expressionvector or 50 ng/ml rhTPO (R & D Systems, Minneapolis, Minn.).Supernatant collected from COS cells transfected with an emptyexpression vector was used as a control for TPO-containing COSsupernatant.

[0088] Single CD34⁺Thy-1⁺Lin⁻ adult bone marrow (ABM) cells werecultured in the presence of 10% control COS supernatant (controls), 10%COS supernatant from COS cells transfected with TPO, 10% TPO COSsupernatant+10 ng/ml IL-3, 10% TPO COS supernatant+25 ng/ml KL, or inthe presence of 10 ng/ml IL-3, IL-6, and LIF, and 25 ng/ml KL.

[0089] Imaging and cell number quantitation. Images of Terasaki wellsingle cell cultures were digitized and stored twice weekly.Magnification was adjusted such that the entire well was visible in eachimage. An inverted Nikon Diaphot microscope and Hamamatsu C2400 NewviconCCD camera were used for image acquisition. Image-1 image analysissoftware and Compaq 66-MHz 486 computer (digitizing hardware) were used(Universal Imaging, Philadelphia, Pa.). A Panasonic LF-7010 rewritableoptical disk drive was used for image storage.

[0090] Cell numbers were determined from the image series by directcounting (<100 cells); or by estimating the proportion of the wellfilled (>100 cells). Average cell number in confluent wells wasdetermined by hemacytometer counting.

[0091] Phenotypic analysis of subconfluent and confluent culture wells.Analysis of CD34 expression on cultured cells by flow cytometry wascarried out as follows. Briefly, single cells which grew to fill anentire Terasaki well with blast cells were individually analyzed for thepercentage of primitive progenitors (CD34⁺ cells). The contents ofsubconfluent Terasaki wells (30-90% filled with blast cells) were pooledfor analysis. Hematopoietic cells were removed from the wells by gentlepipetting, resuspended in 100 μl staining medium, and stained withanti-CD34 (HPCA2)-PE. A pool made from a small aliquot from each of thewells was used for control staining with IgG1-PE. A well was consideredto contain a CD34⁺ population if greater than 1% of the cells showed alevel of fluorescence above that of the isotype control.

[0092] MK Progenitor Fibrin Clot Assay. The serum-depleted fibrin clotassay for colony-forming unit-megakaryocyte (CFU-MK) has been describedpreviously (Bruno et al. (1989) Blood 73:671; Briddell et al. (1989)Blood 74:145). This assay compared the number of CFU-MK present inpopulations of day 0 ABM CD34⁺Thy-1⁺Lin⁻ cells to the number obtainedfrom wells of proliferating blast cells that grew from a singleCD34⁺Thy-1⁺Lin⁻ cell in the presence of TPO. Freshly sorted cells wereplated at 10⁴ cells/ml, or blast populations harvested from Terasakiwells after 3 weeks of culture were plated in the fibrin clot assay inthe presence of IL-3 at 10 ng/ml (Sandoz, Basel, Switzerland), GM-CSF at2 ng/ml, and KL at 100 ng/ml (R&D Systems, Minneapolis, Minn.). Fibrinclots were fixed and analyzed for CD41⁺ colonies at 12 days.

[0093] Results. The effects of TPO on single cell cultures grown in thepresence or absence of other cytokines are shown in Figs. 1A-1E. Theplating efficiency (number of single cells that divided one or moretimes/number of cells plated) and the average cell proliferationachieved by all single cells plated, are also shown. Control COSsupernatant alone did not stimulate proliferation of CD34⁺Thy-1⁺Lin⁻cells (FIG. 1A). TPO-containing COS supernatant supported a platingefficiency of 74% and an average cell production of 454 cells per cell(FIG. 1B). When 25 ng/ml KL was used in combination with TPO COSsupernatant, no augmentation of growth over TPO alone was observed (FIG.1D). TPO plus 10 ng/ml of IL-3 enhanced cell production over TPO aloneapproximately 4-fold (1,681 cells/input cell) (FIG. 1C). This level ofproliferation was similar to that achieved with a combination of fourcytokines (IL-3, IL-6, LIF, and KL) (FIG. 1E). Results were similar whenpurified rhTPO was used (data not shown).

[0094] TPO Expansion of Blast Cells. To determine whether TPO alone, orin combination with other cytokines could support proliferation ofprimitive CD34⁺ cells, single CD34⁺/Thy-1⁺Lin⁻ cells were cultured inthe presence of 5% TPO COS supernatant, TPO+IL-3, TPO+KL,IL-3+IL-6+KL+LIF, and IL-3+IL-6+K1+LIF+TPO. Cells were harvested fromculture wells at 4-6 weeks, and either analyzed individually or pooledwith other wells grown under like conditions. Cell populations werefluorescently stained with anti-CD34 antibody and analyzed by flowcytometry as described above. These populations contained from 0.5-4.4%CD34⁺ cells, average 2.8% for TPO alone compared to 1% for TPO+IL-3,1.2% for TPO+KL, 0.5% for IL-3+IL-6+LIF+KL, and 2.1% forTPO+LIF+KL+IL-3+IL-6. Thus, in these cultures TPO increased theexpansion of CD34⁺ cells, compared to other cytokines tested, withoutdifferentiation into the MK lineage. Blast cell outgrowths did notexpress CD41b by immunocytochemical staining.

[0095] TPO Effect on Growth Morphology of CD34⁺Thy-1+Lin⁻ Cells. Theeffect of TPO on MK differentiation was determined by size, phenotype,and expression of CD41b antigen expression by the methods describedabove.

[0096] Approximately 50% of single CD34⁺Thy-1⁺Lin⁻ cells from ABM whichgrew in the presence of TPO displayed MK differentiation, as determinedby a growth pattern of initial expansion and dispersal of 10-200refractile blast cells over the stromal cell layer, followed bydifferentiation into large 25-50 nm refractile cells. This processoccurred over a 2-3 week period from initial plating. Wells containingcells with this growth morphology were harvested and analyzed for CD41bexpression by immunocytochemical staining. Essentially all hematopoieticcells in these populations stained positively for CD41b. This includedthe large cells as well as those that were still quite small in size.

[0097] To quantitatively demonstrate that TPO was stimulating singlestem cells to produce multiple MK progenitors prior to maturation, thenumber of CFU-MK present in populations of day 0 ABM CD34⁺Thy-1⁺Lin⁻cells were compared to the number obtained from wells of proliferatingblast cells that grew from a single CD34⁺Thy-1⁺Lin⁻ cell in the presenceof TPO. Freshly sorted cells, or blast populations harvested fromTerasaki wells after three weeks of culture were placed in the fibrinclot assay. Colonies containing MKs were enumerated afterimmunofluorescent staining of the fibrin clot cultures with anti-CD41bantibody. In one representative experiment, 5,000 day 0 CD34⁺Thy-1⁺Lin⁻cells produced 13 MK colonies or 0.0026 colonies per cell. Sevenindividual blast cell populations, each arising from one cell, producedan average of 8.3 CFR-MK per cell (range 1-18) in the presence of TPOand IL-3. This represents a 3,200-fold amplification of MK progenitorproduction during culture. These findings indicate that TPO can act onsingle human stem cells and permit commitment to the MK lineage, MKprogenitor expansion, and MX maturation in culture.

EXAMPLE 5 Effects of TPO on Cobblestone Area Forming Cells (CAFC).

[0098] Limiting dilution (LD) cultures were established by platingCD34^(+Thy-)1⁺Lin⁻ cells in Whitlock/Witte medium (50% IMDM (JRHBiosciences), 50% RPMI 1640, 10% FCS, 4×10⁻⁵ M 2-mercaptoethanol, 10 mMHEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, and 4 mM glutamine)at limiting dilution (from 100-0.8 cells/well) on preformed Sys1 stromalmonolayers, as described previously (Murray et al. (1995) supra). Fourcytokine conditions were compared: (1) 5% control mock-transfected COSsupernatant; (2) 5% TPO-containing COS supernatant; (3) 50 ng/ml LIF+10ng/ml IL-6; and (4) 50 ng/ml LIF+10 ng/ml IL-6+5% TPO supernatant. Inone experiment, purified TPO (R&D Systems) was used at 10 ng/ml.Cultures were fed at weekly intervals and scored at week 4 forcobblestone area formation. Wells containing cobblestone areas wereanalyzed for CD34 expression.

[0099] TPO Effect on Size and Frequency of Cobblestone Areas. LDcultures grown with medium alone or with 5% control COS supernatantshowed almost no growth, whereas cultures grown with 5% TPO-containingCOS supernatant showed increased cell growth, including growth ofcobblestone areas. LD cultures grown in the presence of LIF and IL-6,showed cobblestone area formation as observed previously (Murray et al.(1995) supra). However, when TPO was added to LIF and IL-6, thecobblestone areas appeared earlier, at higher frequency (Table 2), andgrew to a much larger size by 4 weeks. When these wells were analyzedfor CD34 ws: expression, 69% contained CD34+cells compared to 38% ofwells grown in LIF and IL-6 alone. Similar results were obtained withpurified rhTPO. In agreement with the findings obtained with single cellculture, TPO increased the expansion of CD34+ cells from primitiveprogenitors cultured at limiting dilution (Table 3).

EXAMPLE 6 Expansion Effects of TPO

[0100] CD34⁺, CD34⁺Lin⁻ or CD34⁺Thy-1⁺Lin⁻ ABM cells were cultured onSyS1 stromal cells in SCCM with the indicated cytokines and analyzed atdays 7 and 11.

[0101] Expansion Effect of TPO on ABM CD34⁺ selected cells. The purityof glycoprotease-selected CD34⁺ cells ranged from 60-93%. TPO aloneinduced a 1.3-fold expansion of CD34⁺ by day 11, at which time a mean of52.8% MKs were observed (Table 4). Wright-Giemsa staining showed bothsmall and large MKs, the latter with multilobulated nuclei, indicativeof polyploidization. Immunocytochemistry confirmed that both small andlarge cells were positive for CD41b expression, showing commitment tothe MK lineage. Although IL-3 is known as an MK stimulatory factor, only3% MKs were detectable at day 11 when CD34⁺ cells were cultured in IL-3alone. A combination of TPO, IL-3, and KL resulted in a 4.7-fold lowerpercentage of MKs at day 11, while the number of total cells increased5.5-fold, producing a similar or slightly higher number of MKs at day11. Results are shown in Table 5.

[0102] Expansion Effect of TPO on ABM CD34⁺Lin⁻ selected cells.CD34⁺Lin⁻ cells (depleted of CD2⁺, CD15⁺, and CD19⁺ cells) purified byFACS sorting had purities from 87% to 96%. In the presence of TPO alone,total cell expansion was greater than that observed with CD34⁺ cells(Table 6). By day 11, there was a 10-fold greater total cell expansionin cells cultured with TPO+IL-3 compared to TPO alone, but theproportion of MKs was approximately 3.5-fold lower (Table 5). TPO+IL-3generated a 2-fold higher number of total MKs by day 11, but the MKswere a minority of the population, the majority of cells appearingmyeloid (Table 5). The addition of GM-CSF to TPO resulted in a 2.8-foldgreater cell expansion by day 11, but the percentage of MKs remainedslightly higher than with TPO+IL-3 (33.7% vs. 20.6%) (Table 5). A higherabsolute number of MKs at day 11 could be produced by addition of eitherIL-3 or GM-CSF to TPO, but at the expense of the purity of the MKpopulation.

[0103] Effect of TPO on ABM CD34⁺Thy-1⁺Lin⁻. Using the original sortgates, purity of the Thy-1⁺ cell subpopulations ranged from 77-90% (mean84%) and of the Thy-1⁻ cell subpopulations from 65-93% (mean 86%). Therewas significant cell expansion from the Thy-1⁺ population and a highpercentage of MKs were consistently observed by day 11 (61.9-77.6%),indicating that CD34⁺Thy-1⁺Lin⁻ stem cells can mature into MKs under thestimulation of TPO alone. The differences in MK potential between theThy-1⁺ and Thy-1⁻ cell subpopulations were not significant (FIGS. 3A &3B). Table 6 summarizes the mean total cell expansion and MK productionof each cell subpopulation at days 7 and 11 under these conditions.

EXAMPLE 7 TPO Stimulates Activation of Quiescent Stem Cells into Cycle.

[0104] To determine if TPO could activate quiescent stem cells intocycle, the effect of TPO on primitive hematopoietic cells which couldefflux Rh123, (CD34⁺Lin⁻ Rhl231^(lo)) was tested. This cell populationwas labeled with the membrane dye PKH26, so that cell division could beobserved by loss of PKH fluorescence. Labeling was performed permanufacturer's instructions (Zynaxis Cell Sciences Inc., Malvern, Pa.).

[0105] The CD34⁺Lin⁻Rh123^(lo) cells labeled with PKH26 dye werecultured for up to 6 days as follows: approximately 10⁴ cells wereseeded in SCCM without stroma at 100 μl/well in round bottom 96 wellplates with either no added cytokines (negative control), or thecytokine indicated (FIGS. 2A-2L). Cells were cultured in the presence ofTPO, KL, or IL-3 alone, TPO and KL, or TPO and IL-3, and then analyzedat day 3 and day 6. Cultures were harvested, stained with anti-CD34-FITCantibody, and analyzed by flow cytometry for CD34 expression versusPKH26 dye retention on days 3 and 6 of culture. To obtain a day 0measurement, an aliquot of cells was taken following staining andincubated overnight at 37° C. to allow complete efflux of the Rh123 dye.Cells were then stained with anti-CD34-FlTC and analyzed by flowcytometry. Cycling of cells was indicated by loss of PKH fluorescence,while differentiation was indicated by loss of CD34 expression.

[0106] FIGS. 2A-2L show a representative summary of 3 experiments usingpurified rhTPO. Similar results were also obtained in two previousexperiments using 10% TPO-containing COS supernatant. Inserum-containing medium, in the absence of added cytokines, the cellsremained in the upper right quadrant (there was no loss of PKH26fluorescence or CD34 expression) showing that the cells remainedquiescent (FIGS. 2A and 2B). By day 3 of culture with rhTPO, about 8% ofRho123^(lo) cells had divided (FIG. 2C). By day 6, 32% of the cells werecycling, and ⅔ of these expressed CD34 (FIG. 2D). At day 3 the effect ofTPO and KL was similar (FIG. 2E), but by day 6 higher percentages ofCD34+ cells were retained in the presence of TPO (FIG. 2F and FIG. 2D).When TPO and KL were combined, activation of cells into cycle by day 3was more than additive, and occurred without loss of CD34 expression(FIG. 2K). There was apparent synergy between KL and TPO by day 6 with69% of the cells entering cycle, however, 62% of cycling cells haddifferentiated to CD34− cells (FIG. 2L). IL-3 alone stimulated thegreatest degree of cycling by day 6, however, the vast majority of thesecells had lost CD34 expression, indicating that IL-3 pusheddifferentiation at the expense of self-renewal (FIG. 2H). With theaddition of TPO to IL-3, more dividing cells retained CD34 expression atdays 3 and 6 than seen with IL-3 alone (FIG. 2J), although fewerdividing cells were CD34⁺ as compared to cultures with both TPO and KL.

EXAMPLE 8 Effect of TPO on Transduction of Stem Cells.

[0107] CD34⁺Thy-1⁺Lin⁻ stem cells are selected from mobilized peripheralblood or ABM as described above. Transduction experiments are conductedby methods known to the art in combination with the present disclosure.In addition to TPO (10-100 ng/ml purified recombinant TPO) thetransduction medium may contain cytokines such as KL, IL-3, IL-6, etc.

[0108] The LN retroviral vector contains the bacterial neo gene as aselectable marker under the control of the Herpes Simplex Virusthymidine kinase (HSV-tk) promoter, with the therapeutic gene ofinterest inserted under the control of the Moloney murine leukemia viruslong terminal repeat (MMLV-LTR) (Miller & Rosman (1989) Biotechniques7:980). The retroviral vector DNA is electroporated (Chu et al. (1987)Nucleic Acids Res. 15:1311) into the amphotropic PA317 packaging cells(Miller & Buttimore (1986) Mol. Cell. Biol. 6:2895) and the cells areselected in 600 mg/ml G418 (Gibco-BRL). Virus from resistant PA317 cellsare used to inoculate GP-E86 cells (Markowitz et al. (1988) J. Virol.62:1120) and G418-resistant cell populations are again established.Virus from GP-E86 cells are used to inoculate PA317 cells at highmultiplicity of infection (>10) and individual G418-resistant PA317 cellclones are isolated. A single clone producing high titer viralsupernatants (>1×10⁶ G418-resistant CFU/ml as determined on NIH 3t3cells) is selected. The producer cells test negative for replicationcompetent retrovirus (RCR) in S+L− assays on PG-4 cells (Haapala et al.(1985) J. Virol. 53:827). Vector-producing cells are grown in DMEM withhigh glucose and 10% FCS. Supernatant is harvested 24 hr after a mediachange on confluent monolayers and stored at −70° C.

[0109] Viral supernatant is diluted 1:1 in 2× transduction medium(Whitlock/Witte medium plus cytokines and 4 ug/ml protamine sulfate)prewarmed to 37° C., and sorted cells are suspended in the diluted viralsupernatant at about 5×10⁵ cells/ml. The same procedure is followed inthe absence of TPO (control). After 4 hrs, the medium is removed fromthe cells, and the cells resuspended in fresh medium (without virus)with or without TPO and/or additional cytokines, and the cellsrepipetted into the original well. The cultures are incubated foranother 20 hrs. The process is repeated for three consecutive days withthe cells incubated in the presence of virus for 4 hr each day. After 72hrs, a viable cell count is performed. Transduction frequency isimproved when the cells are transduced as described above but sample arecentrifuged at 2800 × g at 21° C. during the 4 hr incubation period.

[0110] To determine transduction frequency, 2.5-5×10³ cells from eachtransduction are added to 5 ml of methylcellulose (Stem CellTechnologies) containing the following cytokines: KL, 10 ng/ml; GM-CSF,25 ng/ml; G-CSF, 25 ng/ml; IL-3, 10 ng/ml; and rhEPO, 2 units/ml. 1.1 mlof the cell/cytokine methylcellulose mixture is plated onto four 3 cmgridded plates using a 5 ml syringe and 16.5 gauge needle, and theplates are placed in a 37° C. incubator for 2 weeks. After 14 days,single methylcellulose colonies are picked and suspended in 50 μl LysingBuffer (75 mM KCl, 10 mM Tris-HCl, pH 9.25, 1.5 MM MgCl₂, 0.5% Tween-20,0.5% NP40, 1 mg/ml proteinase K) and PCR used to amplify vector-specificsequences in the transduced cells. PCR products are visualized onethidium bromide agarose gels.

[0111] Long term stromal cultures are used to evaluate gene markings ofmore primitive progenitor cells. Briefly, 10,000 cells from eachtransduction are cultured for 4 weeks on a pre-established monolayer ofmouse stromal cells (AC6.21) in media supplemented with 20 ng/ml LIF and20 ng/ml IL-6. Plates are fed weekly by demi-depletion and growthpositive wells are analyzed by PCR for the presence of the transgene.

[0112] Results. Cells transduced in the presence of TPO will exhibitimproved cell viability relative to cell transduced in the absence ofTPO, hence resulting in higher numbers of transduced stem cells.Transduction frequencies are improved in the presence of IL-3 and KL, inaddition to TPO, which promote cell viability, cell cycling and bindingof amphotropic retrovirus to human hematopoietic stem cells. TABLE 1Single HSC per well culture: analysis of CD34⁺ cells AB week # TPO TPO +IL-3 TPO + KL 3 + 6 + KL + LIF 3 + 6 + KL + LIF + TPO M analyzed wells %CD34⁺ % CD34⁺ % CD34⁺ % CD34⁺ % CD34⁺ 1 4 1 ND 0   ND ND ND 4 1 ND 0  ND ND ND 5 1 ND 1.7 ND ND ND 5 1 ND 2.5 ND ND ND 6 pool 0.5 ND 1.2 1.0ND 2 4 pool 3.5 ND ND ND ND  3′  4 pool 4.4 ND ND 0   2.1 AVE 2.8 1.01.2 0.5 2.1

[0113] TABLE 2 Effect of TPO on CAFC frequency Cytokines added LIF + ABMO TPO LIF + IL-6 IL-6 + TPO 4* 1/1389   1/82  1/44  1/19  5* <1/10,000  1/745  1/228 1/33   6** <1/10,000   1/3060 1/649 1/142

[0114] TABLE 3 Limiting dilution culture: analysis of CD34⁺ cells LIF +ABM TPO (% CD34⁺) LIF + IL-6 (% CD34) IL-6 + TPO (% CD34) 4 13.1 3.014.7 5 4.5 0 4.5 6 4.3 1.8 6.3

[0115] TABLE 4 Effect of TPO and IL-3 with or without KL on ABM CD34⁺selected cells % CD41b⁺ cells total cell no. × 10⁴ Addition day 4 day 7day 11 day 4 day 7 day 11 0 4.4 ± 0.8 4.6 ± 2.2 ND 7.8 ± 1.1 6.0 ± 1.6ND IL-3 6.0 ± 1.4 7.2 ± 1.8 3.0 ± 0.2 12.5 ± 0.5  13.1 ± 1.9  15.6 ±3.2  TPO 20.8 ± 0.0  42.4 ± 7.0  52.8 ± 2.8  11.2 ± 0.2  11.5 ± 0.4 13.2 ± 0.8  TPO + IL-3 20.1 ± 5.3  25.0 ± 3.6  32.0 ± 22.2 18.8  11.0 32.9  IL-3,TPO + KL 6.7 ± 2.6 11.0 ± 1.0  11.3 ± 3.7  34.1 ± 17.6 34.8 ±7.9  72.1 ± 4.3 

[0116] TABLE 5 Effect of TPO with or without IL-3 or GM-CSF on CD34⁺Lin⁻ cells % CD41b⁺ cells total cell no. × 10⁴ AB day day day day dayday M Addition 4 7 11 4 7 11 1 TPO 19.8 70.6 70.1 34.0 28.5 34.2 TPO +IL-3 14.8 14.0 15.2 67.0 95.3 276.4 TPO + GM-CSF 16.0 30.7 34.0 25.052.3 104.6 2 TPO 39.7 43.7 61.4 20.6 28.0 74.2 TPO + IL-3 15.2 17.1 21.090.0 132.0 204.6 TPO + GM-CSF 29.3 31.9 33.3 27.4 88.8 195.4 3 TPO 27.553.5 75.6 15.5 40.3 19.7 TPO + IL-3 22.0 31.2 25.6 23.5 131.6 355.8TPO + IL-3 + GM-CSF 16.5 23.7 16.3 68.6 164.6 117.7

[0117] TABLE 6 Comparison of ABM CD34⁺ Cell Subpopulations Total cellNo. CD41b⁺ % CD41b⁺ Population Additions no. × 10⁴ cells cells Day 7CD34⁺ TPO 11.5 4.9 42.4 TPO + IL-3 11.0 2.8 25.0 CD34⁺Lin⁻ TPO 32.3 18.155.9 TPO + IL-3 120.0 25.0 20.8 CD34⁺Thy⁺Lin⁻ TPO 18.4 9.2 49.6 TPO +IL-3 14.5 7.1 48.8 Day 11 CD34⁺ TPO 13.2 7.0 52.8 TPO + IL-3 32.9 10.532.0 CD34⁺Lin⁻ TPO 42.7 29.5 69.0 TPO + IL-3 279.0 57.5 20.6CD34⁺Thy⁺Lin⁻ TPO 10.5 7.1 67.5 TPO + IL-3 10.5 7.7 73.7

[0118]

1 6 42 base pairs nucleic acid single linear cDNA Inosine 1 GCTATTGCGGCCGCGAATTC GGARGAYYTN NNNTGYTTYT GG 42 38 base pairs nucleic acid singlelinear cDNA Inosine 2 GCTATTCTCG AGATCGATSW CCANTCRCTC CANNNNCC 38 38base pairs nucleic acid single linear cDNA Inosine 3 GCTATTCTCGAGATCGATSW CCANNNRCTC CANNNNCC 38 20 base pairs nucleic acid singlelinear cDNA 4 CGCTGCACCT CTGGGTGAAG 20 21 base pairs nucleic acid singlelinear cDNA 5 AGCAGGGCAG CAGGTTTCTG T 21 21 base pairs nucleic acidsingle linear cDNA 6 GCTGCGCAGC TGCAGCCAGT A 21

What is claimed is:
 1. A method for promoting the survival of a stemcell in culture, comprising culturing said cell in the presence of amyeloproliferative receptor (mpl) ligand, wherein said ligand binds mpland mpl-mediated biological activity is initiated.
 2. The method ofclaim 1, wherein said mpl ligand is thrombopoietin.
 3. The method ofclaim 2, wherein said thrombopoietin is human thrombopoietin.
 4. Themethod of claim 3, wherein said thrombopoietin is recombinant humanthrombopoietin.
 5. The method of claim 1, wherein said cell cultured inthe presence of said mpl ligand is characterized by the capability ofself-renewal and ability to give rise to all hematopoietic celllineages.
 6. The method of claim 1, wherein said cell is a human stemcell.
 7. The method of claim 6, wherein said cell is CD34⁺.
 8. Themethod of claim 6, wherein said cell is CD34⁺Lin⁻.
 9. The method ofclaim 6, wherein said cell is CD34⁺Thy⁺Lin⁻.
 10. The method of claim 6,wherein said cell is CD34⁺+Lin⁻Rho^(lo) or CD34⁺Thy⁺Lin⁻Rho^(lo). 11.The method of claim 4, wherein said recombinant human thrombopoietin ispresent in a concentration of about 1 ng/ml to about 100 ng/ml.
 12. Amethod of expanding a population of stem cells, comprising exposing astem cell to a mpl ligand, wherein said cell proliferates to form anexpanded population of stem cells.
 13. The method of claim 12, whereinsaid mpl ligand is thrombopoietin.
 14. The method of claim 13, whereinsaid thrombopoietin is human thrombopoietin.
 15. The method of claim 14,wherein said thrombopoietin is recombinant human thrombopoietin.
 16. Themethod of claim 12, wherein said expanded cell population ischaracterized by the ability to undergo substantial self-renewal andability to give rise to all hematopoietic cell lineages.
 17. The methodof claim 12, wherein said cells are human stem cells.
 18. The method ofclaim 17, wherein said cell is CD34⁺.
 19. The method of claim 17,wherein said cell is CD34⁺Lin⁻.
 20. The method of claim 17, wherein saidcell is CD34⁺Thy⁺Lin⁻.
 21. The method of claim 17, wherein said cell isCD34⁺Lin⁻Rho^(lo) or CD34⁺Thy⁺Lin⁻Rho^(lo) .
 22. The method of claim 15,wherein said recombinant human thrombopoietin is present in aconcentration of about 1 ng/ml to about 100 ng/ml.
 23. A therapeuticmethod for restoring hematopoietic capability to a human subject, saidmethod comprising the steps of: (a) removing stem cells from a humansubject; (b) expanding said cells in the presence of a mpl ligand toform an expanded population of stem cells from a human subject; and (c)returning said expanded cells to said subject, wherein hematopoieticcapability is restored to said patient.
 24. The method of claim 23,wherein said expanded population of stem cells are characterized by thecapability of self-renewal and ability to give rise to all hematopoieticcell lineages.
 25. The method of claim 23, wherein said mpl ligand isthrombopoietin.
 26. The method of claim 23, wherein said thrombopoietinis human thrombopoietin.
 27. The method of claim 26, wherein saidthrombopoietin is recombinant human thrombopoietin.
 28. The method ofclaim 27, wherein said recombinant human thrombopoietin is present in aconcentration of about 1 ng/ml to about 100 ng/ml.
 29. The method ofclaim 24, wherein said cells are expanded in the presence of one or moreadditional cytokines.
 30. The method of claim 29, wherein said cytokinesare selected from the group consisting of interleukin 3 (IL-3),interleukin 6 (IL-6), leukemia inhibitory factor (LIF), c-kit ligand(KL), granulocyte-macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), and steel factor (Stl).31. The method of claim 30, wherein said cytokine is IL-3.
 32. A methodfor activating a quiescent stem cell to divide, comprising exposing saidquiescent cell to a mpl ligand, wherein said cell is activated todivide.
 33. The method of claim 32, wherein said mpl ligand isthrombopoietin.
 34. The method of claim 33, wherein said thrombopoietinis human thrombopoietin.
 35. The method of claim 34, wherein saidthrombopoietin is recombinant human thrombopoietin.
 36. The method ofclaim 32, wherein said cell is a human stem cell.
 37. The method ofclaim 36, wherein said cell is CD34⁺.
 38. The method of claim 36,wherein said cell is CD34⁺Lin⁻.
 39. The method of claim 36, wherein saidcell is CD34⁺Thy⁺Lin⁻.
 40. The method of claim 36, wherein said cell isCD34⁺Lin⁻Rho^(lo) or CD34⁺Thy⁺Lin⁻Rho^(lo).
 41. The method of claim 32,wherein cells formed from said activated cell are characterized by thecapability of self-renewal and ability to give rise to all hematopoieticcell lineages.
 42. The method of claim 35, wherein said recombinanthuman thrombopoietin is present in the concentration range of about 1ng/ml to about 100 ng/ml.
 43. A method for modifying a stem cell,comprising the steps of: (a) inserting a foreign gene into a viralvector; (b) culturing a quiescent stem cell in the presence of a mplligand, wherein said cell is activated to divide; and (c) exposing saidactivated cell to said viral vector, wherein said foreign gene isintegrated into the DNA of said stem cell.
 44. The method of claim 43,wherein said mpl ligand is thrombopoietin.
 45. The method of claim 44,wherein said thrombopoietin is human thrombopoietin.
 46. The method ofclaim 45, wherein said thrombopoietin is recombinant humanthrombopoietin.
 47. A method for providing gene therapy to a subject,comprising providing the modified stem cell of claim 43 to a subject inneed thereof.
 48. The method of claim 31, wherein said foreign geneencodes a protein selected from the group consisting of the mdr1 geneproduct, adenosine deaminase, glucocerebrosidase, β-globin, Factor VIII,Factor IX, mdr related protein, T-cell receptors, and cytokines.
 49. Themethod of claim 31, wherein said foreign gene is an antisense orribozyme sequence.
 50. The method of claim 43, wherein saidthrombopoietin is a thrombopoietin mimetic.
 51. The method of claim 43,wherein said viral vector is a retroviral vector.
 52. The method ofclaim 43, further comprising the steps of: transplanting said final cellpopulation into a recipient to provide long term hematopoieticreconstitution.
 53. The method of claim 52, wherein said initialhematopoietic cell population is obtained from said recipient.
 54. Themethod of claim 52, further comprising the step of selecting CD34⁺ cellsfrom said final population prior to said transplanting step.
 55. Themethod of claim 54, wherein said selecting step further selects cellsfrom said final population that are Thy-1⁺.
 56. The method of claim 45,wherein said human thrombopoietin is present in a concentration of about1 ng/ml to about 100 ng/ml.
 57. The method of claim 50, wherein saidthrombopoietin mimetic is present in a concentration of about 1 ng/ml toabout 100 ng/ml.
 58. The method of claim 43, wherein said medium furthercomprises at least one cytokine selected from the group consisting ofinterleukin 3 (IL-3), interleukin 6 (IL-6), leukemia inhibitory factor(LIF), c-kit ligand (KL), granulocyte-macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF) and fetalliver kinase 2 (FLK-2) ligand.
 59. The method of claim 43, wherein saidinitial population of cells is selected for positive expression of CD34prior to said culturing step.
 60. The method of claim 43, wherein saidgene of interest encodes a protein selected from the group consisting ofthe mdr1 gene product, adenosine deaminase, glucocerebrosidase,β-globin, Factor VII, Factor IX, mdr related protein, T-cell receptors,and cytokines.
 61. The method of claim 43, wherein said gene of interestis an antisense or ribozyme sequence.
 62. The method of claim 43,wherein said retrovirus is an amphitropic retrovirus.
 63. A method forgenetically modifying a population of human hematopoietic stem cells,comprising the steps of: culturing in vitro an initial hematopoieticcell population comprising human CD34⁺Thy-1⁺ hematopoietic stem cells ina medium comprising a myeloproliferative receptor (mpl) ligand, whereinsaid population of hematopoietic stem cells proliferates to expand thenumber of CD34⁺ cells in a final cell population; transducing saidhematopoietic cell population with a viral vector comprising a gene ofinterest, wherein said final cell population comprises humanhematopoietic stem cells that have been genetically modified byintegration of said gene of interest into the cells; and transplantingsaid final cell population into a recipient to provide long termhematopoietic reconstitution.
 64. The method of claim 63, wherein saidinitial hematopoietic cell population is obtained from said recipient.65. The method of claim 64, wherein said gene of interest is anantisense or ribozyme sequence.